Introduction

This pipeline is used to generate the base-pairing map from deep mutational sequencing data of RNA ribozyme, with the method introduced in the paper "Accurate inference of the full base-pairing structure of RNA by deep mutational scanning and covariation-induced deviation of activity".

Requirement

Before running this pipline, make sure these programs were correctly installed.

SeqPrep (https://github.com/jstjohn/SeqPrep)

python 2.7.15

unpigz 2.4

gsplit 8.29

pigz 2.4

samtools 0.0.18

java 1.7.0_85

Usage

bash run.sh $DNAFILE1 $DNAFILE2 $RNAFILE1 $RNAFILE2 $FASTAFILE $OUTPUTPATH

Arguments:

$DNAFILE1: first read DNA sequencing file, in gziped fastq format (fq.gz)

$DNAFILE2: second read DNA sequencing file, in gziped fastq format (fq.gz)

$RNAFILE1: first read RNA sequencing file, in gziped fastq format (fq.gz)

$RNAFILE2: second read RNA sequencing file, in gziped fastq format (fq.gz)

$FASTAFILE: base sequence file in fasta format (ATGC sequence)

$OUTPUTPATH: all output files will be written here, empty file folder is recommended

Outputs:

var.count: uncleaved and cleaved read number of each variant

var.ra: organized relative activity of each variant

var.pos.ra: organized relative activity of all mutants of each position pair

var.msa_RA_0.5: sequence alignment of variants with relative activity higher than 0.5

pred.mtx: ps score matrix

pred.ss: 100 predicted secondary structure in the bracket format with a consensus prediction

MSA(Multiple sequence alignment) files:

cp.var.msa_RA_0.5: MSA of CPEB3 used to perform covariation analysis.
tw.var.msa_RA_0.5: MSA of CPEB3 used to perform covariation analysis.